Method for mechanical and hydrodynamic microfluidic transfection and apparatus therefor

ABSTRACT

Methods for introducing exogenous material into a cell are provided, which include exposing the cell to a transient decrease in pressure in the presence of the exogenous material. Also provided are devices for performing the method of the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/AU2015/050748, filed 26 Nov. 2015, which claims the benefit ofAustralian Provisional Application No. 2015900021, filed 7 Jan. 2015.The entire contents of each of the above-identified applications ishereby incorporated by reference herein.

FIELD OF THE INVENTION

The invention relates to a method for introducing exogenous materialinto a cell, comprising exposing the cell to a transient decrease inpressure in the presence of the exogenous material. The transientdecrease in pressure is preferably coupled with an unsteady flow ofliquid in which the cell and exogenous material are present. Inparticular, the invention relates to transfection of mammalian cells.

BACKGROUND OF THE INVENTION

Any discussion of the prior art throughout the specification should inno way be considered an admission that such prior art is widely known orforms part of common general knowledge in the field.

The introduction of exogenous material such as small organic molecules,proteins and nucleic acids into cells in vitro and in vivo is crucialfor the progression of research and development of therapies, as well astherapeutic delivery strategies.

For example, the introduction of fluorescently tagged proteins intocells allows real time analysis of the trafficking of the proteinsthroughout the cells, which may also assist in the identification ofprotein interactions during clinically important stages of a disease, orin response to specific triggers. Introducing putative small organicmolecule drugs, which do not naturally cross cell membranes effectively,into cells during drug development can be informative on the activity ofsaid drugs prior to diverting valuable time and effort towardsdeveloping delivery vehicles for these drugs.

The introduction of nucleic acids into cells is a critical step in celltherapy manufacturing, where expression vectors encoding genes aredelivered across the cell membrane into the cytoplasm to effectivelyengineer live cells that can be used as therapeutic agents. By way ofexample, cell therapy may be used to induce an individual's own immunesystem to attack cancer cells or evade a virus, such as HIV. Cancer andHIV are of particular relevance from a global health perspective giventheir prevalence in the population with an estimated 35 million HIVpatients in 2013 and 14 million new cancer cases in 2012. Utilisingcell-derived gene therapy as part of a global health strategy requires acell therapy manufacturing method capable of reproducibly producingsufficient quantities of product to potentially treat tens of millionsof patients per annum at the appropriate price point and under currentGood Manufacturing Practice in accordance with regulatory standards.

Accordingly, the ability to introduce exogenous material and inparticular nucleic acids into cells in a quick and efficient manner isboth a valuable research tool and a useful component of a therapeuticstrategy.

There are several known methods for introducing agents into cells, withthe choice of method generally being determined by the type of cell, thelevel of efficiency required, the size of the molecule being introducedand the number of cells available.

Although the terms may be used interchangeably, the introduction ofagents such as nucleic acids into eukaryotic cells is generally referredto as “transfection”, whereas the introduction of nucleic acid intoprokaryotic cells is generally referred to as “transformation”.Transfection and transformation methods may be conveniently separatedinto three categories, namely, chemical, physical and viral-basedmethods.

Chemical methods of transfection employ reagents such as cationiclipids, calcium phosphate, cationic polymers and dendrimer molecules toessentially package the nucleic acids for delivery into the cell.However, many of these methods are not applicable to all cell types.Moreover, they can be compromised by pH fluctuations or salts/phosphatesin the cell media. Due to the requirement for packaging of nucleic acidsin some of these methods, the size of the nucleic acid molecules thatcan be accommodated may be limited. Further, chemical transfectionmethods can require use of reagents that are expensive and/or toxic tocells in high concentrations and/or the method may only achievelow/inconsistent transfection efficiencies.

Conventional physical methods used to transfect eukaryotic cells includethe use of magnetic nanoparticles, electroporation, bolistic particledelivery and microinjection. However, these methods tend to be quiteharsh on the cells, often resulting in high mortality rates. Thesemethods may also require immobilised cells, expensive equipment and/or agreater degree of technical skill on the part of the person performingthe method. For example, in some electroporation methods, suspendedcells are first permeabilised, followed by the application of anelectric field to facilitate active delivery of charged exogenousmaterial. Hence these techniques require specialised equipment andconsumables for permeabilising the membranes of the cells and applyingthe electrical field.

Viral-based transfection methods rely on viral vectors includinglentiviral, adenoviral and retroviral vectors for the delivery ofnucleic acids into a cell, where the nucleic acids may be expressed athigh levels by virtue of a viral promoter. These viral-based methods areexpected to prove useful for the effective treatment of cancers of thelymphatic and haematopoietic systems, and for HIV therapeutics. However,due to variable transfection efficiencies, the cost of manufacturingviral vectors for these types of therapeutics is in the order ofthousands of dollars per patient. Further, this method can be bothlabour intensive and prone to manufacturing issues if the process is notautomated.

The introduction of exogenous material, and in particular, nucleicacids, into prokaryotic cells is also an important aspect for themanufacture of biologics during therapeutic drug development and indeed,research in general. Transformation of bacterial cell lines withexogenous nucleic acids for the recombinant production of valuablemolecules such as biologic-based pharmaceuticals (so calledbiopharmaceuticals) can be achieved by various methods includingchemical transformation and electroporation. However, these methods mayrequire that the cells be made “competent” prior to transformation(e.g., by inducing high cell density and/or nutritional limitation whichswitches on a set of genes), they may not be applicable to all celltypes and/or they may result in high levels of cell mortality.

Consequently, there is a need for a fast and efficient method ofintroducing exogenous material into a range of cell types that overcomesone or more of the difficulties of the known methods. Preferably, themethod would deliver an acceptable level of cell viability and it wouldbe cost-effective.

It is an object of the invention to overcome or ameliorate at least oneof the disadvantages of the prior art, or to provide a usefulalternative.

It will be appreciated that reference herein to “preferred” or“preferably” is intended as exemplary only.

SUMMARY OF THE INVENTION

The limitations associated with the introduction of exogenous materialinto cells are often related to the toxicity and/or expense associatedwith the reagents and devices used for physical, viral and chemicaltransfection and transformation methods. Further, many transfectionmethods are not adaptable to high-throughput applications partly becausethey require significant human intervention throughout the process andlarge volumes of cells to compensate for the low transfectionefficiencies and/or cell viabilities. Indeed, human intervention isoften the source of the inconsistencies associated with the transfectionefficiencies of methods that are heavily reliant on technicians. Theintroduction of even low levels of imprecision by humans can havesignificant adverse effects on delivery efficiency, cell viabilityand/or repeatability. See, for example Mitsuyasu et al. (Mitsuyasu R.T., et al. (2009). Phase 2 gene therapy trial of an anti-HIV ribozyme inautologous CD34+ cells. Nature Medicine, 15(3):285-292) wherein viralvectors were used to transfect CD34+ hematopoietic progenitor cellsresulting in 54±17% (mean±standard deviation) delivery efficienciesacross 38 patients (n=38).

It has been surprisingly found by the inventor that, when exposed to atransient decrease in pressure, cells are susceptible to the uptake ofexogenous material.

Without wishing to be bound by theory, the transient decrease inpressure most likely permeabilises the cell membrane without lysing thecell. A relatively sudden and temporary pressure drop across the cellmembrane, whereby the intracellular pressure is greater than theextracellular pressure, may result in the temporary formation of poresin the membrane allowing for the introduction of the exogenous material.

Accordingly in a first aspect of the invention, there is provided amethod for introducing an exogenous material into a cell, comprisingexposing the cell to a transient decrease in pressure in the presence ofsaid exogenous material to thereby introduce said exogenous materialinto said cell. The transient decrease in pressure does not result inthe cell being lysed although in certain embodiments it may be renderednon-viable. The skilled addressee will understand that when theinvention is applied to a population of cells, some of the cells in thepopulation may be lysed.

Preferably the cell is viable after being exposed to the transientdecrease in pressure.

Preferably, the cell is selected from the group consisting of abacterial cell, a mammalian cell, a yeast cell, a plant cell and aninsect cell.

In certain preferred embodiments, the cell is a mammalian cell. In otherpreferred embodiments, the cell is a bacterial cell. In yet otherpreferred embodiments, the cell is a yeast cell. In further preferredembodiments, the cell is an insect cell. In yet further embodiments, thecell is a plant cell.

Preferably the exogenous material is selected from the group consistingof small organic molecule, nucleic acid, nucleotides, proteins,peptides, amino acids, lipids, polysaccharides, viruses, quantum dots,carbon nanotubes, radionuclide, magnetic bead, nanoparticles, goldparticles, monosaccharides, vitamins and steroids.

Preferably the nucleic acids are selected from the group consisting ofPNA, DNA, RNA, mRNA, miRNA and siRNA.

Preferably the DNA is a plasmid.

Preferably the plasmid is an expression vector.

Preferably the expression vector expresses PNA, DNA, RNA, miRNA, siRNAor protein.

Preferably the expression vector is a viral vector.

Preferably the viral vector is a lentiviral vector or retroviral vector.

Preferably the expression vector is a bacterial artificial chromosome(BAC) or a yeast artificial chromosome (YAC).

Preferably the exogenous material is introduced into the cytoplasm ofthe cell.

Preferably the exogenous material is introduced into the nucleus of thecell. In these preferred embodiments, the cell is a mammalian cell, ayeast cell, a gamete (e.g., a sperm cell or an ovum cell) or an insectcell. More preferably, the cell is a mammalian cell.

Preferably the transient decrease in pressure is a decrease of at least10 kPa.

Preferably the transient decrease in pressure is a decrease of at least100 kPa.

Preferably the transient decrease in pressure is a decrease of at least500 kPa.

Preferably the transient decrease in pressure is a decrease of at least1000 kPa.

Preferably the cell is exposed to said transient decrease in pressure inthe presence of said exogenous material for at least 10 nanoseconds.

Preferably the cell is exposed to said transient decrease in pressure inthe presence of said exogenous material for at least 100 nanoseconds.

Preferably the cell is exposed to said transient decrease in pressure inthe presence of said exogenous material for at least 1 microsecond.

Preferably the cell is exposed to said transient decrease in pressure inthe presence of said exogenous material for no more than 1 millisecond.

Preferably the exogenous material and said cell are in a liquid whenbeing exposed to said transient decrease in pressure.

Preferably the cell is exposed to said transient decrease in pressurewithin an enclosed channel with dimensions configured to allow a flow ofsaid liquid comprising said exogenous material and said celltherethrough.

Preferably the flow of said liquid in said channel has a fluctuatingvelocity.

Preferably the flow has a minimum peak velocity of at least 1 meter persecond.

Preferably the flow has a minimum peak velocity of at least 5 meters persecond.

Preferably the flow has a maximum peak velocity of no more than 50meters per second.

Preferably the flow has a maximum peak velocity of no more than 100meters per second.

Preferably the channel is configured to influence the flow of saidliquid such that there are one or more regions within the channel wherethe flow of said liquid is laminar, and/or one or more regions withinthe channel where the flow of said liquid is creeping, and/or one ormore regions within the channel where the flow of said liquid isunsteady.

Preferably the object Reynolds number (Re_(o)) of the flow of the liquidaround a flow diverter in at least one of said regions within thechannel is sufficient to induce unsteady flow.

Preferably the object Reynolds number (Re_(o)) is at least 40.

Preferably the object Reynolds number (Re_(o)) is no more than 2000.

Preferably the flow of liquid is influenced by one or more flowdiverters within said channel.

Preferably the one or more regions within the channel where the flow ofsaid liquid is unsteady is downstream of said flow diverter.

Preferably the cell is exposed to said transient decrease in pressuredownstream of said flow diverter.

Preferably the flow diverter is an obstacle placed within said enclosedchannel.

Preferably the obstacle is a post. More preferably, the post iscylindrical.

Preferably the obstacle is positioned in said channel such that saidcell must pass through a gap with a width and height, or diameter, atleast 1.01× the minimum diameter of said cell when flowing through saidchannel.

Preferably the gap has a width and height, or diameter, at least 1.01×the minimum diameter of said cell.

Preferably the gap has a width and height, or diameter, at least 2× theminimum diameter of said cell.

Preferably the gap has a width and height, or diameter, at least 10× theminimum diameter of said cell.

Preferably the gap has a width and height, or diameter, at least 100×the minimum diameter of said cell.

Preferably the obstacle has a maximum width of 10 nanometers.

Preferably the obstacle has a maximum width of 20 micrometers.

Preferably the obstacle has a maximum width of 100 micrometers.

Preferably the obstacle has a maximum width of 1 millimetre.

There are numerous advantages to adapting high-throughput methods forintroducing exogenous material into cells to meet the demands oflarge-scale manufacturing. For example, devices such as microfluidicdevices, can be suitably designed and operated to expose cells to one ormore transient decreases in pressure in the presence of exogenousmaterial. Advantageously, the devices may be manufactured from simpleplastics at very low cost, potentially in the range of only a fewdollars per device.

Accordingly, in a second aspect of the invention, there is provided adevice for use in a method for introducing an exogenous material into acell in a liquid, comprising;

-   -   an at least partially enclosed channel with dimensions        configured to allow the flow of said cell and an exogenous        material suspended in a liquid therethrough; and    -   one or more flow diverters within said channel;    -   wherein the flow diverter results in at least one region of        decreased pressure immediately downstream of said flow diverter.

Preferably the region of decreased pressure occurs in at least oneregion of unsteady flow immediately downstream of said flow diverter.

Preferably the device is a microfluidic device.

Preferably the device is configured according to FIG. 4.

Preferably the device is configured according to FIG. 5.

Preferably the device is configured according to FIG. 6.

Preferably the device is configured according to FIG. 7.

Preferably the device is configured according to FIG. 8.

Preferably the device is configured according to FIG. 9.

Preferably, the device is configured according to FIG. 10.

Preferably the device is used in a method for introducing exogenousmaterial into a cell in a liquid according to any one of previousaspects.

Accordingly, in a third aspect of the invention, there is provided acell comprising an exogenous material produced according to any one ofthe previous aspects.

Accordingly, in a fourth aspect of the invention, there provided is acell suspension comprising a cell of the fourth aspect.

Accordingly, in a fifth aspect of the invention, there is provided apharmaceutical composition comprising a cell of the third aspect or acell suspension of the fourth aspect, and a pharmaceutically acceptablediluent, cryopreservant, carrier or excipient.

Accordingly, in a sixth aspect of the invention, there is provided a kitcomprising a device of the second aspect.

The invention relates to the introduction of exogenous material into acell. As used herein, the term “exogenous” means any material thatexists outside of the cell prior to the cell being exposed to thetransient decrease in pressure in the presence of the exogenousmaterial. It will be understood that the term “exogenous” relates tomaterial that has been developed, grown or originated outside the cell.The exogenous material may be naturally occurring or synthetic. In thecontext of the present application, the term “naturally occurring”insofar as it relates to a material means any material that exists innature, and may include biologically active substances. The naturallyoccurring materials may be modified in ways that do not naturally occurin nature and is suitably isolated from nature by techniques as known inthe art. In the context of the present application, the term “synthetic”is meant not naturally occurring, but made through human technicalintervention. In the context of synthetic proteins and nucleic acids,this encompasses molecules produced by recombinant, chemical syntheticor combinatorial techniques as are well known in the art. The syntheticmaterial may be an imitation of a naturally occurring material, or maynot be analogous to a material that exists in nature.

The exogenous material may be biologically active in the cell into whichthe material is introduced. Alternatively, the exogenous material mayhave no detectable effect on the cell after it is introduced.

The cell may be any cell with a cell membrane or cell wall that may betemporarily permeabilised when said cell is exposed to a transientdecrease in pressure. The cell may or may not be viable before or afterbeing exposed to the transient decrease in pressure in the method of theinvention. The cell may or may not be senescent. As the method of theinvention may be a passive method of introducing exogenous material intoa cell, it would be understood that it is not essential that the methodof the invention be performed on cells that are viable and/or activelydividing. For example, in the event the exogenous material was beingintroduced into the cell to identify a particular organelle in thecytoplasm, the method of the invention could be performed on dead cells(cells that are no longer capable of metabolising). In another example,if the exogenous material being introduced into the cell was a selectivemarker for cell death, the method of the invention could be performed ona mixture of live and dead cells.

In particular embodiments of the invention, the cell is a bacterialcell, a mammalian cell, a yeast cell, a gamete cell (e.g., a sperm cellor an ovum cell), a plant cell or an insect cell. It will be appreciatedthat the invention also contemplates a progenitor cell and in particulara stem cell and more preferably, a hematopoetic stem cell or mesenchymalstem cell. The cell may be in culture, extracted from tissue samplesand/or immortalised. It will be appreciated that in those embodimentsthat contemplate a plant cell, the cell wall is completely or partiallyremoved to form a protoplast, prior to treatment according to themethods of the present invention. The cell may be from a primary cultureor may from a continuous (secondary) culture. The cell may be derivedfrom any tissue type. The cell may or may not be terminallydifferentiated. Suitably, the cell is an isolated cell. By “isolated” ismeant material that is substantially or essentially free from componentsthat normally accompany it in its native state, or from componentspresent during its production when purified or produced by syntheticmeans. Thus, the term “isolated” also includes within its scope purifiedor synthetic material.

As will be appreciated by a person of skill in the art, preferredstarting cell densities may be dependent on the cell type and/orexogenous material. In preferred embodiments of the invention and inparticular preferred embodiments that relate to mammalian cells, thestarting cell density is between about 2 million cells per mL to about10 million cell per mL, and all integers in between.

In the context of “introducing exogenous material into a cell” asrecited herein, the term “introducing” means that the exogenous materialis delivered into, travels into or transfers into at least theouter-most barrier of a cell i.e., into the cell wall or cell membrane.The exogenous material may travel beyond the outer-most barrier of acell, and pass through the cell wall or cell membrane to enter thecytoplasmic region of the cell. The exogenous material may travel intoorganelles within the cell. Specifically, the exogenous material maytravel into the nucleus of the cell.

In embodiments of the invention, the exogenous material being introducedinto the cell is selected from the group consisting of a small organicmolecules, a nucleic acid, a nucleotide, an oligonucleotide, a protein,a peptide, an amino acid, a lipid, a polysaccharide, a quantum dot, ananoparticle, a monosaccharide, a gold particle, a vitamin and asteroid, and combinations thereof. The exogenous material need not havea net charge.

Preferably, the nucleic acid is selected from the group consisting ofPNA, DNA, RNA, miRNA and siRNA, and combinations thereof. Preferably,the DNA is an oligonucleotide or plasmid.

In particular embodiments of the invention, the plasmid is an expressionvector. An expression vector may be either self-replicatingextra-chromosomal vector such as a plasmid, or a vector that integratesinto a host genome. As used herein, the term “vector” refers to anymolecule used as a vehicle to assist in the delivery or expression of anucleic acid in a cell. Preferably, the vector expresses DNA, RNA,miRNA, siRNA or protein. By “vector” is meant a polynucleotide molecule,suitably a DNA molecule derived, for example, from a plasmid,bacteriophage, virus, yeast or higher order eukaryote including plant,vertebrate or invertebrate animal, into which a polynucleotide can beinserted or cloned. A vector preferably contains one or more uniquerestriction sites and can be capable of autonomous replication in adefined host cell including a target cell or tissue or a progenitor cellor tissue thereof, or be integratable with the genome of the definedhost such that the cloned sequence is reproducible. Accordingly, thevector can be an autonomously replicating vector, i.e., a vector thatexists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a linear or closedcircular plasmid, an extrachromosomal element, a minichromosome, or anartificial chromosome. The vector can contain any means for assuringself-replication. Alternatively, the vector can be one which, whenintroduced into the host cell, is integrated into the genome andreplicated together with the chromosome(s) into which it has beenintegrated. A vector system can comprise a single vector or plasmid, twoor more vectors or plasmids, which together contain the total DNA to beintroduced into the genome of the host cell, or a transposon. The choiceof the vector will typically depend on the compatibility of the vectorwith the host cell into which the vector is to be introduced. In someembodiments, the vector is a viral or viral-derived vector, which isoperably functional in vertebrate or invertebrate animal and suitablymammalian cells. Such vector may be derived from a poxvirus, alentivirus, a retrovirus, an adenovirus or yeast. The vector can alsoinclude a selection marker such as an antibiotic resistance gene thatcan be used for selection of suitable transformants. Examples of suchresistance genes are known to those of skill in the art and include thenptII gene that confers resistance to the antibiotics kanamycin and G418(Geneticin®) and the hph gene, which confers resistance to theantibiotic hygromycin B.

In other embodiments of the invention, the vector is a viral vector,preferably a lentiviral vector or a retroviral vector. The vector mayalso be a bacterial artificial chromosome or a yeast artificialchromosome.

In the context of the invention, the term “lysed” means that the cellwall/cell membrane of said cell is sufficiently compromised such thatthe bulk of the content of the cell is no longer contained within thecell wall/cell membrane and the cell is thus rendered non-viable.However, even if a cell is not lysed, it need not necessarily be viableto be useful in the invention. The skilled addressee would appreciatethat there may be circumstances when it would be desirable to transfecta cell with exogenous material, without the requirement that theresultant transfected cell be viable, provided the cell is not lysed.For example, in the event the exogenous material was a marker orantibody designed to bind and indicate the location or expressionprofile of a particular protein in a cell, the cell's viability may notbe a determining factor of the assay outcome. In a further example, themethod of the invention may result in cells that are not lysed, but arenecrotic and still of interest.

In particular embodiments of the invention, the cell is viable afterbeing exposed to the transient decrease in pressure in the method of theinvention. It would be understood that a “viable” cell is one that iscapable of cellular metabolism and/or cell division. A cell that iscapable of cellular metabolism is one that is capable of degradingmolecules and releasing energy (generally referred to as catabolism),making molecules (such as polysaccharides, lipids, nucleic acids andproteins) and/or using energy (generally referred to anabolism). Aviable cell may be one that is capable of cellular metabolism, but ispermanently in the G₀ phase of the cell cycle and not capable of celldivision.

The method of the invention may be used to transfect a population ofcells, and within this population of cells, some cells may be lysed (andhence not viable), some cells may not be lysed but may not be viable,while others may be viable.

As used herein, the term “decrease in pressure” insofar as it relates toexposure of a cell to such a decrease means the cell is exposed to azone of pressure that is relatively lower than the pressure immediatelysurrounding the zone. The pressure in the zone may be uniform or mayhave localised regions of varied pressure provided these localisedregions still have a pressure that is lower relative to the pressuresurrounding the zone. The pressure surrounding the zone may be uniformor may have localised regions of varied pressure provided theselocalised regions have a pressure that is higher relative to the zone.

By “pressure” is meant the force per unit area exerted by a substance onits surroundings as is known in the art. The SI unit of pressure is thepascal (Pa). Other commonly used units for the measurement of pressureinclude kilopascals (kPa), pound forces/square inch (PSI), millimetresof mercury (mmHg), millibars (mbar), and atmospheres (atm) air pressure.Pressure specifically relating to a vacuum may be measured in torrs(Torr). In the present application when the term “kPa” is used, itrefers to gauge pressure, not absolute pressure where a gauge pressureof 0 kPa refers to an absolute pressure of 101.325 kPa.

The transient decrease in pressure may be defined in the context of thepressure differential between a zone of lower pressure relative to thepressure of a surrounding zone. The transient decrease in pressure mayalso be defined in the context of the minimum pressure in the zone oflower pressure and the maximum pressure in the surrounding zones. Forexample, if the minimum pressure in the zone of lower pressure was −10kPa and the maximum pressure in the surrounding zone was 100 kPa, thenthe pressure differential would be 110 kPa. In another example, if theminimum pressure in the zone of lower pressure was 20 kPa and themaximum pressure in the surrounding zone was 500 kPa, then the pressuredifferential would be 480 kPa. In a further example, the pressuredifferential between the zone of lower pressure and the surrounding zonemay be 200 kPa, which could be the result of the minimum pressure in thezone of lower pressure being in the range of −100 kPa to 1000 kPa andthe maximum pressure in the surrounding zone being in the range of 100kPa to 1200 kPa. In yet another example, the pressure differentialbetween the zone of lower pressure and the surrounding zone may be 50kPa, which could be the result of the minimum pressure in the zone oflower pressure being in the range of 0 kPa to 150 kPa and the maximumpressure in the surrounding zone being in the range of 50 kPa to 200kPa.

The maximum and minimum pressure that can be applied to any one celltype will be apparent to the competent skilled addressee. At pressuresthat are too low, the efficiency of the method may be compromised and atpressures that are too high, the cells may rupture. The optimum pressuredifferential may be identified for a particular cell by reference to theexamples of the present application and through routine experimentation.

Preferably, the transient decrease in pressure that the cell is exposedto in the presence of the exogenous material is a decrease of at least10 kPa, at least 100 kPa, at least 500 kPa or at least 1000 kPa. Incertain embodiments, the transient decrease in pressure is a decrease inpressure (kPa) of at least 15, at least 20, at least 25, at least 30, atleast 35, at least 40, at least 45, at least 50, at least 60, at least70, at least 80, at least 90, at least 100, at least 150, at least 200,at least 250, at least 300, at least 350, at least 400, at least 450, atleast 500, at least 550, at least 600, at least 650, at least 700, atleast 750, at least 800, at least 850, at least 900, at least 950 or atleast 1000.

The term “transient” in the context of a decrease in pressure means thatthe decrease in pressure occurs temporarily, in that after the cell isexposed to the decreased pressure, the pressure that the cell is exposedto afterwards will be of higher pressure. In some embodiments of theinvention, the transient decrease in pressure means that the cells areexposed to a minimum pressure reached during a particular exposure forat least 10 nanoseconds, but no more than 1 millisecond. It would beunderstood that this time is not inclusive of the time between when thecell is exposed to a maximum pressure in a surrounding zone to themoment when the cell is exposed to a minimum pressure in a zone of lowerpressure relative to the pressure of the surrounding zone. This time isalso not inclusive of the time between when the cell is exposed to aminimum pressure in a zone of lower pressure to the moment when the cellis exposed to a maximum pressure in a surrounding zone.

The time that any one cell type can be exposed to the transient decreasein pressure will be determinable by the competent skilled addressee.Exposures that are too long may result in inefficiencies, whileexposures that are too short may not allow for the introduction of theexogenous material into the cell. The optimum exposure times can bedetermined for a particular cell by reference to the examples of thepresent application and through routine experimentation.

Preferably, the cell is exposed to a transient decrease in pressure inthe presence of the exogenous material for at least 10 nanoseconds, atleast 100 nanoseconds, at least 1 microsecond or at least 1 millisecond.In certain embodiments of the invention, the cell is exposed to atransient decrease in pressure for at least 15 nanoseconds, at least 20nanoseconds, at least 25 nanoseconds, at least 30 nanoseconds, at least35 nanoseconds, at least 40 nanoseconds, at least 45 nanoseconds, atleast 50 nanoseconds, at least 60 nanoseconds, at least 70 nanoseconds,at least 80 nanoseconds, at least 90 nanoseconds, at least 100nanoseconds, at least 150 nanoseconds, at least 200 nanoseconds, atleast 250 nanoseconds, at least 300 nanoseconds, at least 350nanoseconds, at least 400 nanoseconds, at least 450 nanoseconds, atleast 500 nanoseconds, at least 550 nanoseconds, at least 600nanoseconds, at least 650 nanoseconds, at least 700 nanoseconds, atleast 750 nanoseconds, at least 800 nanoseconds, at least 850nanoseconds at least 900 nanoseconds, at least 950 nanoseconds, at least100 microseconds, at least 200 microseconds, at least 300 microseconds,at least 400 microseconds, at least 500 microseconds, at least 600microseconds, at least 700 microseconds, at least 800 microseconds, orat least 900 microseconds.

By “transient” is also meant that the decrease in pressure occursrelatively rapidly, in that the time between when the cell is exposed toa maximum pressure in a surrounding zone to the moment when the cell isexposed to a minimum pressure in a zone of relatively lower pressure isless than 1 second. In particular embodiments of the invention, the timebetween when the cell is exposed to a maximum pressure in a surroundingzone to the moment when the cell is exposed to a minimum pressure in azone of relatively lower pressure is less than 1 millisecond. Similarly,once the cell is exposed to a minimum pressure in a zone of relativelylower pressure, the time between the cell being exposed to this minimumpressure and the time the cell is exposed to a maximum pressure in asurrounding zone is less than 10 seconds, 100 seconds or 1 minute. Inthe invention, the time between the cell being exposed to this minimumpressure and the time the cell is exposed to a maximum pressure in asurrounding zone is sufficient to permeabilise the membrane but not lysethe cell.

The cell may be exposed to more than one transient decrease in pressurein the presence of the exogenous material when a method of the inventionis performed. In some embodiments, the cell may be exposed to more thanone transient decrease in pressure wherein the transient decreases inpressure are the same or different in terms of the pressure differentialbetween the zone of relatively lower pressure and a surrounding zone. Inother embodiments of the invention, the pressure differential thatdefines the transient decreases may be due to the same or differentminimum pressure in the zone of relatively lower pressure. The pressuredifferential that defines the transient decreases may also be due to thesame or different maximum pressure in the surrounding zones.

For example, the cell may be exposed to a transient decrease in pressurewherein the pressure differential is 10 kPa, followed by a secondtransient decrease in pressure wherein the pressure differential is also10 kPa. The first transient decrease in pressure of 10 kPa may be theresult of the minimum pressure in the zone of relatively lower pressurebeing 50 kPa and the maximum pressure in the surrounding zone being 40kPa, while the second transient decrease in pressure of 10 kPa may bethe result of the minimum pressure in the zone of relatively lowerpressure being 20 kPa and the maximum pressure in the surrounding zonebeing 30 kPa.

In another example, the cell may be exposed to a transient decrease inpressure wherein the pressure differential is 300 kPa, followed by asecond transient decrease in pressure wherein the pressure differentialis 80 kPa. The first transient decrease in pressure of 300 kPa may bethe result of the minimum pressure in the zone of relatively lowerpressure being 100 kPa and the maximum pressure in the surrounding zonebeing 400 kPa, while the second transient decrease in pressure of 300kPa may be the result of the minimum pressure in the zone of relativelylower pressure being −50 kPa and the maximum pressure in the surroundingzone being 250 kPa.

In embodiments of the invention, the cell is exposed to the transientdecrease in pressure in the presence of the exogenous material when bothare in a liquid. The liquid may be any liquid that does not ordinarilyresult in lysis of the cell and, in some embodiments of the invention,is capable of maintaining the viability of the cell for the duration ofthe method. Preferably, the exogenous material would be soluble in,capable of being suspended in, or would be dispersible in, the liquid.For example, the liquid may be a cell growth media, or a buffered salinesolution, such as phosphate buffered saline, or tris buffered saline.The liquid may be blood, plasma or serum or another bodily fluid, suchas whole blood, cord marrow, bone marrow or adipose-derived fluids. Theblood or bodily fluid may be fractionated, separated and/or diluted forimproved processing. Although the fluid may contain agents or chemicalsthat promote the introduction of the exogenous material into the cell,the liquid need not necessarily contain any additional agents orchemicals to facilitate the introduction of the exogenous material intothe cells. For example, in certain embodiments of the invention, theliquid does not comprise any additional cationic lipids, cationicpolymers, calcium ions (for example, in the form of calcium chloride orcalcium phosphate), magnesium ions (for example, in the form ofmagnesium chloride) or dendrimers. It would be understood that many ofthese chemicals and agents are toxic to cells, and the absence, orsubstantial absence of added amounts of these chemicals or agents in theliquid used in the method of the invention may prevent unwanted celllysis or cell death when performing the method of the invention.

By “additional” is meant any additional amount of the chemical or agentin addition to what may normally and/or naturally be present in theliquid. For example, many bodily fluids, such as blood, may naturallycomprise calcium ions, but in particular embodiments of the invention,no calcium phosphate would be added to the blood before being used asthe liquid in a method of the invention. In another example, a cellgrowth media may normally comprise magnesium ions, but in particularembodiments of the invention, no magnesium chloride would be added tothe growth media before being used as the liquid in the method of theinvention.

In preferred embodiments of the invention, the cell is exposed to thetransient decrease in pressure in a liquid within a channel, preferablyan enclosed channel, with dimensions configured to allow the flow of theliquid comprising the exogenous material and the cell therethrough. Inthe context of the invention, by “channel” is meant any component with alength and two or more ends, with a hollow space extending the length ofthe component that allows the flow of a liquid through the hollow space,and through openings at the two or more ends. The dimensions of thechannel need only be configured to allow the flow of a relevant celltype in said liquid. A cross-section of the channel may have any shape.The channel should comprise at least some enclosed sections but it isnot necessarily sealed along the entirety of its length as long as thereare areas within the channel in which the required pressure changes mayoccur.

It would be understood that the flow of the liquid would essentially befrom one end of the channel to the other, and the direction of the flowwould determine the orientation of what was “upstream” and “downstream”.

Flow through the channel may be caused by various means, including butnot limited to hydrostatic pressure, hydrodynamic pressure and/orelectro-osmotic flow. The flow of the liquid may be driven by a pressuresource, including but not limited to, a pressure pump, a gas cylinder, acompressor pump, a vacuum pump, a syringe, a syringe pump, a peristalticpump, a piston, a capillary pump, a heart, a muscle or gravity.

The pressure source used to generate the flow of liquid through thechannel would preferably provide steady-state flow such as creeping flowor laminar flow, as the liquid enters the channel. The skilled addresseewould understand that creeping flow refers to a flow of liquid where theinertial forces of the liquid are significantly lower than the viscousforces of the liquid. Laminar flow refers to a flow of liquid where theinertial forces within the liquid are greater than or equal to theviscous forces of the liquid, but not great enough to inducetransitional or turbulent flow in the liquid.

The flow of the liquid through the channel will have a velocity, andthis velocity may be influenced by factors including, but not limitedto, the configuration of the channel, the strength and nature of thepressure source, the viscosity of the liquid, the cell type and celldensity in the liquid and/or the nature and amount of the exogenousmaterial.

In preferred embodiments of the invention, the velocity of the liquidfluctuates as it flows through the channel, and the fluctuating velocitymay be defined in terms of a maximum velocity and a minimum velocity ofthe liquid as it flows through the channel. The velocity of the liquidmay fluctuate between a particular maximum and minimum velocity as theliquid flows through the channel. Preferably, the fluctuating velocityof the liquid flowing through the channel has a minimum peak velocity of1 meter per second, or more preferably, 5 meters per second. In otherpreferred embodiments of the invention, the fluctuating velocity of theliquid flowing through the channel has a maximum velocity of 10 metersper second, a maximum velocity of 20 meters per second, a maximumvelocity of 30 meters per second, a maximum velocity of 40 meters persecond, a maximum velocity of 50 meters per second, a maximum velocityof 60 meters per second, a maximum velocity of 70 meters per second, amaximum velocity of 80 meters per second, a maximum velocity of 90meters per second or a maximum velocity of 100 meters per second.Accordingly, it would be understood that the peak velocity of the liquidflowing through the channel may fluctuate between a range of 1 meter persecond to 100 meters per second.

As the liquid flows through the channel, as well as the flow having afluctuating velocity, the type of flow may change. For example, the flowmay alternate between being laminar flow, creeping flow and unsteadyflow where unsteady flow refers to a laminar vortex street, atransitional vortex street, a turbulent vortex street, transitional flowor turbulent flow. The skilled addressee would understand the differencebetween creeping flow, laminar flow and unsteady flow. In particularembodiments of the invention, the channel is configured to influence theflow of the liquid such that there are one or more regions within thechannel where the flow of the liquid is laminar, one or more regionswithin the channel where the flow of the liquid is creeping, and one ormore regions within the channel where the flow of the liquid isunsteady.

The type of flow may be estimated by calculating two different Reynoldsnumbers: one for a particular flow through an enclosed channel (Re_(c))and/or region between a flow diverter, and one for flow around on object(Re_(c)). For example, for creeping flow, Re_(c) is significantly lessthan unity (Re_(c)<<1) and for laminar flow, Re_(c) is between unity andapproximately two thousand (1<Re_(c)<2000). For example, for unsteadyflow around an object, Re_(o) is greater than approximately forty(Re_(o)>40) or sufficient to induce unsteady flow. Re_(c) may be definedas the ratio of the mean liquid velocity (ū) and the hydraulic diameter(D_(h)), to the kinematic viscosity (v) of the liquid, and this equationis defined below. For wide channels where the width is significantlygreater than the height (or vice versa), D_(h) may be substituted withtwice the length of the shorter distance. When calculating the channelReynolds number (Re_(c)) of flow between posts, this equation is usedand the hydraulic diameter of the channel (D_(h)) refers to thehydraulic diameter of the channel between posts (FIG. 1) and the meanliquid velocity (ū) refers to the mean velocity between posts.Re _(c) =ūD _(h) /v

In certain embodiments of the invention, the channel Reynolds number(Re_(c)) of the flow of the liquid in at least one of the regions withinthe channel where the flow of the liquid is laminar is at least 100, butno more than 2000. In certain embodiments, the channel Reynolds number(Re_(c)) of the flow of the liquid in at least one of the regions withinthe channel where the flow of the liquid is laminar is at least 100, atleast 200, at least 300, at least 400, at least 500, at least 600, atleast 700, at least 800, at least 900, at least 1000, at least 1100, atleast 1200, at least 1300, at least 1400, at least 1500, at least 1600,at least 1700, at least 1800, at least 1900 or about 2000.

In preferred embodiments of the invention, the flow of liquid isinfluenced by one or more flow diverters within the channel. As usedherein, a “flow diverter” is any element or member that results in theflow of the liquid through the channel being diverted in a localisedregion resulting in a localised region of decreased pressure, optionallycoupled with unsteady flow.

In particular embodiments, the flow diverter is an obstacle placed inthe channel. The term “obstacle” relates to any object placed within thechannel that results in the flow of the liquid to be diverted around theobject, resulting in a localised region of decreased pressure ordecreased pressure coupled with unsteady flow substantially immediatelydownstream of the obstacle. The obstacle must be such that the cell canproceed through the channel beyond the obstacle. In preferredembodiments, the obstacle may extend outwards from an inner surface ofthe channel in a direction generally perpendicular to the length of thechannel. The obstacle may extend from one side of the length of thechannel to another side. Alternatively, the obstacle may only partiallyextend from one side of the length of the channel.

In certain embodiments of the invention, the obstacle has a widthbetween 10 nanometers and 1 millimeter and all integer widths inbetween. In preferred embodiments, the obstacle has a width of more than50 nanometers, more than 100 nanometers, more than 500 nanometers, morethan 800 nanometers, more than 1 micrometer, more than 10 micrometers,more than 50 micrometers, more than 100 micrometers, more than 200micrometers, more than 500 micrometers, more than 800 micrometers orabout 1 millimeter. In preferred embodiments the obstacle has a width ofless than 1 millimeter, less than 800 micrometers, less than 500micrometers, less than 200 micrometers, less than 100 micrometers, lessthan 50 micrometers, less than 10 micrometers, less than 1 micrometer,less than 800 nanometers, less than 500 nanometers, less than 100micrometers or less than 50 nanometers. In particularly preferredembodiments, the obstacle width is about 20 μm.

In particular embodiments, the obstacle is a post. In the context of theinvention, an obstacle that is a “post” may be an obstacle that is aprism with a height greater than or equal to its greatest width. Thepost may be cylindrical, triangular, square, polygonal, wing-shaped orany other shape and the specific shape may be selected to tune thetransient decrease in pressure for a given channel Reynolds number(Re_(c)) and/or unsteady flow for a given object Reynolds number(Re_(o)). In particularly preferred embodiments, the post iscylindrical.

The mean velocity of the flow through the channel and directly upstreamof a flow diverter may be such that a transient decrease in pressure isinduced just downstream of the flow diverter or a transient decrease inpressure and a localised region of unsteady flow is induced justdownstream of the flow diverter. In embodiments of the invention whereinthe flow diverter is a post, appropriate inducing mean upstreamvelocities may be calculated using the Reynolds number for the flow ofthe liquid around the post (Re_(o)). For the flow of a liquid around acylindrical post, an Re_(o) of at least forty (Re_(o)≥40) is likely tobe required to induce unsteady flow downstream of the post. For otherpost geometries, the Re_(o) required to generate unsteady flow willdepend on the specific shape of post and the mean upstream liquidvelocity would need to be tuned to create (1) a transient decrease inpressure of sufficient magnitude; or (2) unsteady flow and a transientdecrease in pressure of sufficient magnitude. Re_(o) is defined as theratio of the mean upstream velocity (ū) and the characteristic length ofthe post (l) to the kinematic viscosity (v) of the fluid as shown below.Re _(o) =ūl/v

In certain embodiments of the invention, the object Reynolds number(Re_(o)) of the flow of the liquid in at least one of the regions withinthe channel where the flow of the liquid is unsteady is at least 40, butno more than 2000. In certain embodiments, the object Reynolds number(Re_(o)) of the flow of the liquid in at least one of the regions withinthe channel where the flow of the liquid is unsteady is at least 40, atleast 50, at least 60, at least 70, at least 80, at least 90, at least100, at least 200, at least 300, at least 400, at least 500, at least600, at least 700, at least 800, at least 900, at least 1000, at least1100, at least 1200, at least 1300, at least 1400, at least 1500, atleast 1600, at least 1700, at least 1800, at least 1900 or about 2000.In preferred embodiments the object Reynolds number (Re_(o)) of the flowof the liquid in at least one of the regions within the channel wherethe flow of the liquid is unsteady is less than 50, less than 60, lessthan 70, less than 80, less than 90, less than 100, less than 200, lessthan 300, less than 400, less than 500, less than 600, less than 700,less than 800, less than 900, less than 1000, less than 1100, less than1200, less than 1300, less than 1400, less than 1500, less than 1600,less than 1700, less than 1800, less than 1900 or less than 2000.

Although not wishing to be bound by any particular theory, inembodiments of the invention wherein there is a localised region ofunsteady flow substantially immediately downstream of a flow diverter,the cells may be exposed to a two-way increase in pressure as follows:(1) a localised increase in pressure caused by the unsteady flow; and(2) an increase in pressure following the transient decrease inpressure. This may create a pressure drop across the permeabilised cellmembrane where the extracellular pressure is greater than theintracellular pressure and it may facilitate the active delivery ofexogenous material near the cell membrane and/or exogenous material maybe introduced into the cell by, for example, diffusion or flow from thelocal extracellular environment to the cytosol.

The positioning of any obstacle within the channel will generally resultin regions of the channel with passages or gaps that the cells must passthrough that are smaller in height, width or diameter than other regionsof the channel. However, it would be understood that these regions withthe smaller dimensions must still be configured such that the liquidcomprising the exogenous material and the cell is still able to flowtherethrough. In order to facilitate this, any gap created in thechannel by an obstacle that is required to allow the liquid comprisingthe exogenous material and the cell to flow therethrough, wouldpreferably be at least 1.01× the minimum diameter of said cell. It wouldbe understood that cells are generally not perfectly spherical, and assuch, the minimum diameter of a cell would be the minimum width of acell when the shortest cross section is taken through the cell.

In particular embodiments of the invention, the gap has a width andheight, or diameter, at least 1.01× the minimum diameter of the cell. Inother embodiments, the gap has a width and height, or diameter, at least2×, 5×, 10× or 100× the minimum diameter of the cell.

The invention also relates to devices for introducing exogenous materialinto a cell in a liquid comprising a channel with dimensions configuredto allow the flow of said cell and exogenous material suspended in aliquid therethrough; and one or more flow diverter within said channel;wherein the flow diverter results in at least one region of decreasedpressure immediately downstream of said flow diverter.

In particular embodiments of the invention, the device is a microfluidicdevice.

Suitably, the pharmaceutical compositions of the invention comprise anappropriate pharmaceutically acceptable carrier, diluent cryoprotectant,or excipient. Preferably the pharmaceutically acceptable carrier,diluent or excipient is suitable for administration to mammals and morepreferably, to humans. By “pharmaceutically acceptable carrier” is meanta pharmaceutical vehicle comprised of a material that is notbiologically or otherwise undesirable, i.e., the material may beadministered to a subject along with the selected active agent withoutcausing any or a substantial adverse reaction. Carriers may includeexcipients and other additives such as diluents, detergents, coloringagents, wetting or emulsifying agents, pH buffering agents,preservatives, and the like. Useful reference describingpharmaceutically acceptable carriers, diluents and excipients isRemington's Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991)and Remington: The Science and Practice of Pharmacy (PharmaceuticalPress, London, 22^(nd) Edition, 2012) which is incorporated herein byreference.

In embodiments that contemplate a cell suspension, it will be understoodthat the liquid of the suspension may be the liquid in a method of theinvention was performed on, with or without additional components. Acell suspension may also refer to a dessicated or alternatively, afreeze-dried formulation as is understood in the art.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, preferred methods andmaterials are described. For the purposes of the invention, thefollowing terms are defined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element. As used herein, the use of the singular includes the plural(and vice versa) unless specifically stated otherwise.

By “about” is meant a quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length that varies by asmuch 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a referencequantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length.

In the context of the invention, the words “comprise”, “comprising” andthe like are to be construed in their inclusive, as opposed to theirexclusive, sense, that is in the sense of “including, but not limitedto”.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an overview of unit geometry of a device according to anembodiment of the invention.

FIG. 2 shows an overview of the pressure changes that occur duringsimulations of an embodiment of a method of the invention.

FIG. 3 is an overview of experimental transfection data taken byfluorescent microscopy (left) and optical microscopy (right) at amagnification of 20×, wherein HEK293 cells were transfected with pcDNA3.1 in accordance with the parameters shown in Table 1.

FIG. 4 is a schematic diagram of a microfluidic device containing threecolumns of posts (n_(c)=3) and four rows of posts (n_(r)=4) according toone embodiment of the invention.

FIG. 5 is a schematic diagram of a microfluidic device containing threecolumns of posts (n_(c)=3) and four rows of posts (n_(r)=4) according toanother embodiment of the invention.

FIG. 6 is a schematic diagram of a microfluidic device containing threecolumns of posts (n_(c)=3) and four rows of posts (n_(r)=4) according toyet another embodiment of the invention.

FIG. 7 is a schematic diagram of a microfluidic device containing threecolumns of posts (n_(c)=3) and four rows of posts (n_(r)=4) according toyet a further embodiment of the invention.

FIG. 8 is a sectional view of a device design according to a preferredembodiment of the invention. Panel A is an exploded view of the arraydesign (3× magnification) whilst Panel B is an exploded view of the postdesign present on the array (9× magnification).

FIG. 9 is a sectional view of a device design according to anotherpreferred embodiment of the invention. Panel A is an exploded view ofthe array design (3× magnification) whilst Panel B is an exploded viewof the post design present on the array (9× magnification).

FIG. 10 is a sectional view according to yet another preferredembodiment of the invention. Panel A is an exploded view of the arraydesign (3× magnification) whilst Panel B is an exploded view of the postdesign present on the array (9× magnification). The diagrammaticrepresentations in FIG. 10 are not drawn to scale.

Some figures contain color representations or entities. Colorillustrations are available from the Applicant upon request or from anappropriate Patent Office. A fee may be imposed if obtained from thePatent Office.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

Although the invention has been described with reference to certainembodiments detailed herein, other embodiments can achieve the same orsimilar results. Variations and modifications of the invention will beobvious to those skilled in the art and the invention is intended tocover all such modifications and equivalents.

The invention is further described by the following non-limitingexamples.

EXAMPLE 1

A method and device of the invention was assessed by transfecting a cellmodel with pcDNA 3.1 (Invitrogen™), which expresses green fluorescentprotein (GFP). The device used was a microfluidic device configured withan array of posts, wherein the gap between posts was greater than thecell diameter.

Methods

Simulation & Analysis

Simulation by computation fluid dynamics (CFD) with the finite-volumemethod was employed to examine the microenvironment around the gapsbetween posts for the parameters shown in Table 1 and the devicegeometry shown in FIG. 1. FIG. 1 shows an overview of unit geometry of adevice according to the invention, where a liquid with a velocity (Q)enters the enclosed channel at an inlet, along with cells with adiameter (d_(c)) suspended in a liquid, wherein dc that is less than thegap width (g). Other variables represent post diameter (d_(p)), channelwidth (w) and channel height (h). Three-dimensional geometry was builtin SolidWorks with an inlet length of 100 μm and an outlet length of1000 μm for solving purposes. A structured mesh was constructed in ICEMCFD 14.5 and element quality was checked using the determinant, angleand aspect ratio. Solutions were obtained using ANSYS FLUENT 14.5 on aWindows 7 Enterprise 64-bit computer with an Intel Core i5-3470 CPU at3.20 GHz and 16.0 GB of RAM. A coupled pressure-velocity solver was usedto solve for velocity, pressure and shear stress contours. The channelReynolds number (Rec) was calculated according to the parameters inTable 1, using the interior dimensions of the constriction and theequation below:Rec=2ρQ/μ(g+h)

The boundary conditions for the channel top, bottom and walls defined byposts were set to no slip. Boundary conditions for fluidic sidewallswere set to zero shear. Inlet velocity was defined by an averagevelocity and the outlet was set to a zero pressure boundary condition.

TABLE 1 Summary of experimental parameters. Parameter Value Cell typeHEK293 Cell density 1 × 10⁵ cells ml⁻¹ Cell diameter (d_(c)) 13 μmFloure scent molecule pcDNA 3.1 (GFP plasmid) Molecule density 890 ngml⁻¹ Channel height (h) 40 μm Channel width (w) 400 μm  Post diameter(d_(p)) 20 μm Post gap (g) 30 μm Row shift (s)  0 μm Row pitch (p_(r))50 μm Rows (n_(r)) 9 posts Column pitch (p_(c)) N/A Columns pitch(n_(c)) 1 post Media viscosity (μ) 7.987 × 10⁻⁴ Pa s Media density (ρ)1,006 kg m⁻³ Flow rate (Q) 5 ml min⁻¹ Channel Reynolds number (Re_(c))375 Object Reynolds number (Re_(o)) 131 Oscillating frequency (ƒ) 44.4kHzTransfection

Master moulds of microfluidic devices were fabricated using standardphotolithography techniques, while devices were replicated using softlithography and bonded to glass using oxygen plasma. An overview of thedevice design and transfection parameters are shown in FIG. 1 and Table1 respectively.

HEK293 (Human embryonic kidney 293) cells were suspended in cell mediaat a density of 1×10⁵ cells ml⁻¹, and pcDNA 3.1 GFP plasmids were seededat a density of 890 ng ml⁻¹. This suspension was loaded into a syringeand pumped into the microfluidic device with a flow rate of 5 ml min⁻¹,which corresponds to a Re_(c) of 375 at the gap between posts as theflow cell contained an array of 8 units separated with a 20 μm diameterpost with a gap between posts of 30 μm. This also corresponds to aRe_(o) of 131. Subsequently, cells were incubated for a period of 6 daysthen imaged via both fluorescent and optical microscopy to examine greenfluorescent protein gene expression.

Results: Simulation & Analysis

Simulations indicated a high-pressure region occurs just upstream of theposts in the device of FIG. 1 and a decreased pressure region occursjust downstream of the posts. This means that as the cells flow past theposts, they are exposed a sudden and transient decrease in pressure.Additionally, these simulations were run as transient to determine ifunsteady flow occurs. FIG. 2 shows an overview of the pressure changesthat occur during simulations of an embodiment of the method of theinvention showing (a) pressure contours, (b) velocity magnitude, (c)x-direction velocity of the liquid, which can be used to approximatecell velocity and (d) the y-direction liquid velocity with alternatingjets due to unsteady flow. As shown in FIG. 2, there are significantflow velocities in the x-direction perpendicular to bulk flow in they-direction in the enclosed channel, meaning unsteady flow is occurring.

According to the simulations, as a cell passed through a gap betweenposts positioned in the enclosed channel of the device, it moves from asurrounding zone with a localised pressure of 43.5 kPa, is exposed to atransient decrease in pressure of 94.3 kPa as it enters a zone ofrelatively lower pressure, which has a minimum pressure of 50.8 kPa. Themagnitude of the transient decrease in pressure may vary depending onthe phase of the oscillation. Additionally, cell velocity in the liquidis estimated to be 15 m s⁻¹ during this transient decrease in pressure,which occurs over a distance of approximately 40 μm for a transientdecrease in pressure (dP/dt) of −35.4×10⁶ kPa s⁻¹, wherein dP/dt is thechange is pressure (dP) over change in time (dr). dP is change inpressure between local maxima and local minima, dt is change in timebetween local maxima pressure and local minima.

Subsequently, the unsteady flow conditions subject the cell rapidlychanging flow velocities in the direction orthogonal (y-direction) tothe direction the cell is moving (x-direction), as shown in FIG. 2d ,where peak y-direction velocity ranges from −8.5 m s⁻¹ to 8.3 m s⁻¹ andthese localised unsteady flows are approximately 20 μm wide. Themagnitude of the localised unsteady flow decays as the cell moves awayfrom the posts and decays completely after approximately 500 μm—in thisspace a cell is pulsed by approximately 5 unsteady flows with a velocitymagnitude of between 3.4 m s⁻¹ and −8.5 m s⁻¹. During this period cellvelocities are estimated to be between 10 m s⁻¹ and 15 m s−1 andunsteady flow widths are approximately 20 μm, indicating pulse timesrange between 2.0 μs and 1.3 μs. After the cell is pulsed with atransient decrease in pressure and the unsteady flow, the pressureincreases to the same pressure as the outlet as the cell exits themicrofluidic device or as the cell moves away from the gap.

The simulations suggest the exposure to unsteady flow creates a pressuredrop across the cell membrane where the local extracellular pressure isgreater than the local intracellular pressure, thereby facilitatingactive (mechanical) delivery. Additionally, the increase in pressure asthe cell moves towards the device outlet suitably to facilitates activedelivery due to the pressure drop across the permeabilised cellmembrane.

Transfection

As shown in FIG. 3, the use of a transient decrease in pressure andunsteady flow conditions through a post array can be used to transfectHEK293 cells with pcDNA 3.1 GFP plasmids. The images in the top row aretaken from the same field of view as the images in the bottom row.Bright spots in images on the left-hand side of the panel representHEK293 cells successfully transfected with pcDNA 3.1, which were viableand continued to express green fluorescent protein 6 days aftertransfection.

The simulations allow for unsteady flow, and preliminary simulationswere used to determine which velocity was the most appropriate forcalculating Re_(o) based on the transition from laminar flow conditionsto unsteady flow conditions. The velocity of the liquid used forcalculating Re_(o) varies in the literature, however, previoussimulations confirm the mean upstream velocity is the appropriatevelocity. For example, for liquid flow around a cylindrical post theobject Reynolds number (Re_(o)) may be calculated with the equationbelow:Re _(o) =ρv∞d/μwhere v∞ refers to the velocity of a bulk liquid relative to thecylindrical post, and in this case, the mean upstream velocity of theliquid before the cylindrical post. This would be 8.68 m s⁻¹ for theparameters shown in Table 1, resulting in an Re_(o) of 131.2.

In order to estimate the frequency of oscillation, the correlation shownbelow is used as it applies to flow of liquid around cylindrical posts,where the Re_(o) is between 40 and 190. The Strouhal number (Sr) (adimensionless number used to describe unsteady flow) maybe calculatedfrom Re_(o) with the following correlations for flow around acylindrical post:Sr=0.2665−1.018/√Re _(o) for (40<Re _(o)<190)

This calculation results in a Sr of 0.17, and the frequency ofoscillation (f) may be calculated with the equation below with theliquid velocity (v) and characteristic length (L), which is equal to thediameter of the post (d_(p)):f=Sr v/d _(p)

For the parameters described above, it is estimated the unsteady flowoscillates at a frequency of 44.4 kHz. These unsteady oscillations arealso known to induce structural vibrations within the posts themselves.Thus, it is believed cells may be exposed to a transient decrease inpressure, 44.4 kHz unsteady flow along with induced structuralvibrations.

Laminar flow (Re>>1) between one or more flow diverters, such as (butnot limited to) posts, may be used to create a region of transientlydecreased pressure substantially immediately downstream of the posts.This may be used to suddenly and temporarily decrease ambient pressuresurrounding a cell as it flows past the posts of devices such as thoseshown in FIGS. 1, and 4 to 10. Additionally, if Re_(o)>40 then theseflow characteristic are known to induce unsteady flow, and in theexample describe above, this pulses cells with (1) a transient decreasein pressure and (2) unsteady flow. Moreover, this may be achieved usingchannel dimensions that are greater than cell dimensions (g>d_(c)) tomitigate clogging issues. This facilitates the transfer of exogenousmaterial across the cell membrane and into the cytoplasm. According toPawell et al (Pawell R. S., et al. (2013). Limits of parabolic flowtheory in microfluidic particle separation: a computational study. ASME4th International Conference on Micro/Nanoscale Heat and Mass Transfer,Hong Kong, China. December 11-14.) for channel Reynolds numbers above100 (Re_(c)>100) between posts this creates a region of negligible shearstress. That is, under these conditions, membrane permeabilisation isnot due to shear stress, which indicates that transfection may be aresult of the transient decrease in pressure and unsteady flowconditions along with any conditions induced by the unsteady flow, suchas structural vibrations in the posts, as observed by Renfer et al.(Renfer A., et al. (2013) Vortex shedding from confined micropostarrays. Microfluidics and Nanofluidics. 15(2):231-242).

EXAMPLE 2

Experiments were performed to investigate the extent to which themagnitude and duration of the decrease in pressure affects transfection.

Methods

Two cultures of HEK293 cells were seeded at a density of 100,000 cellsml⁻¹, wherein culture 1 contained HEK293 cells and green fluorescentprotein pcDNA 3.1 seeded at a density of approximately 900 ng 10⁻⁵cells, and culture 2 contained HEK293 cells and 25-based pairoligonucleotides seeded at a density of 100 ng 10⁻⁵ cells. Both cultureswere placed in a vacuum dessicator and the pressure decreased to −95 kPaover the course of 2 minutes. The vacuum was then released and returnedto atmospheric pressure over the course of 10 seconds.

Results & Discussion

This experiment using a prolonged decrease in pressure resulted in niltransfection. No cells expressed GFP and the co-localisation ofoligonucleotides and cells was negligible. When compared to Example 1,the magnitude of the decrease in pressure was substantially greater (a95 kPa decrease, as opposed to a 20 kPa decrease in Example 1). However,the rate of decrease was substantially slower. In Example 1, it isestimated that the rate in which the transient decrease in pressureoccurs (dP/dt) is −35.4×10⁶ kPa s⁻¹. In the present example, the dP/dtis approximately −0.8 kPa s⁻¹. Accordingly, dP/dt may play a role inpermeabilising the cell membrane as the cell membrane is gas permeable,such that if dP/dt is too low gas transfer will occur naturally throughthe cell membrane without permeabilising the membrane. Once dP/dt issufficient, it is thought that the physical properties of cell membranewill not be able to accommodate for rapid gas transfer from theintracellular environment to the extracellular environment. Thus, thecell membrane may be stressed to a point where pores form, therebyallowing the introduction of exogenous material into the cell.

EXAMPLE 3

FIG. 4 is a schematic diagram of a microfluidic device containing threecolumns of posts (n_(c)=3) and four rows of posts (n_(r)=4). The arrayis configured such that the diameter of the posts (d_(p)) is equal tothe gap (g) between posts (d_(p)=g), and the posts for each column isshifted a sufficient distance to bifurcate flow from the previous gapwhere the shift distance (s) is equal to half row pitch (s=p_(r)/2) andthe column pitch (p_(c)) is equal to the row pitch (p_(c)=p_(r)). Thewidth of the channel, number of columns (n_(c)) and number of rows(n_(r)) will vary for each specific device using this or a similardesign.

FIG. 5 is a schematic diagram of a microfluidic device containing threecolumns of posts (n_(c)=3) and four rows of posts (n_(r)=4). The arrayis configured such that the diameter of the posts (d_(p)) is greaterthan the gap between posts (g), and the posts for each column is shifteda sufficient distance to bifurcate flow from the previous gap where theshift distance (s) is equal to half row pitch (s=p_(r)/2) and the columnpitch (p_(c)) is equal to the row pitch (p_(c)=p_(r)). The width of thechannel, number of columns (n_(c)) and number of rows (n_(r)) will varyfor each specific device using this or a similar design.

FIG. 6 is a schematic diagram of a microfluidic device containing threecolumns of posts (n_(c)=3) and four rows of posts (n_(r)=4). The arrayis configured such that the diameter of the posts (d_(p)) is less thanthe gap (g) between posts (d<g), and the posts for each column isshifted slightly from the previous gap where the shift distance (s) isless than half the row pitch (s<p_(r)/2) and the column pitch (p_(c)) isgreater to the row pitch (p_(c)>p_(r)). The width of the channel, numberof columns (n_(c)) and number of rows (n_(r)) will vary for eachspecific device using this or a similar design.

FIG. 7 is a schematic diagram of a microfluidic device containing threecolumns of posts (n_(c)=3) and four rows of posts (nr=4). The array isconfigured such that the diameter of the posts (d_(p)) is less than thegap (g) between posts (d_(p)<g), and the posts for each column isshifted slightly from the previous gap where the shift distance (s) isless than half the row pitch (s<p_(r)/2) and the shift directionswitches with each row. The column pitch (p_(c)) is greater to the rowpitch (pc>p_(r)). The width of the channel, number of columns (n_(c))and number of rows (n_(r)) will vary for each specific device using thisor a similar design.

Preferred embodiments of a device design of the invention are depictedin FIGS. 8, 9 and 10. Both embodiments contain a single inlet and asingle outlet with different internal post figurations that areparticularly shown in Panels A and B of each figure. In theseembodiments, all substrates are fused silica with a substrate thickness(t_(s)) of 700 μm. The unit includes a lid with 2 through-holes, eachhaving a diameter (D_(h)) of 700 μm. The lid and substrate bond strengthor burst pressure should be greater than (>>) 10 atm and once bonded,the total device has a thickness (t_(d)) of 1.40 mm. The devicefootprint of 4.80 mm×9.80 mm accounts for a dicing width of 200 μm. Itis contemplated that 7×6 devices are arrayed across 70 mm×30 mm jig fora total of 42 devices. The bottom piece of the device is deep reactiveion-etched fused silica, bonded to a laser-machined fused silica waferusing a bulk material bond. For the embodiments shown in FIGS. 8 and 9,the substrate etched is to create a channel having a width of 1.5 mm,length of 7.5 mm and a depth of 40.0 μm. For the embodiment shown inFIG. 10, the substrate etched is to create a channel having a width of0.6 mm, length of 5.5 mm and a depth of 40.0 μm.

According to the embodiment shown in FIG. 8, the array design (Panel A)includes thirty (30) posts in the x-direction (n_(x)) and one (1) row ofposts in the y-direction (n_(y)) with an array pitch of 50.0 μm in thex-direction (P_(x); otherwise referred to as the column pitch p_(c)). Inthis embodiment, the post design as shown in Panel B, is configured suchthat the diameter of the posts (d_(p)=20 μm) is less than the 30.0 μmgap of between the posts (gap=P_(x)−d_(p)) that is present in thisembodiment.

According to the embodiment shown in FIG. 9, the array design (Panel A)includes thirty (30) posts in the x-direction (n_(x)) and three (3) rowsof posts in the y-direction (n_(y)) with an array pitch of 50.0 μm inthe x-direction (P_(x); otherwise referred to as the column pitch p_(c))and 750 μm in the y-direction (Py; otherwise referred to as the rowpitch p_(r)). In this embodiment, the post design as shown in Panel B,is configured such that the diameter of the posts (d_(p)=20 μm) is lessthan the 30.0 μm gap of between the posts (gap=P_(x)−d_(p)) that ispresent in this embodiment.

According to the embodiment shown in FIG. 10, the array design (Panel A)includes twelve (12) posts in the x-direction (n_(x)) and three (3) rowsof posts in the y-direction (n_(y)) with an array pitch of 50.0 μm inthe x-direction (P_(x); otherwise referred to as the column pitch p_(c))and 500 μm in the y-direction (Py; otherwise referred to as the rowpitch p_(r)). In this embodiment, the post design as shown in Panel B,is configured such that the diameter of the posts (d_(p)=20 μm) is lessthan the 30.0 μm gap of between the posts (gap=P_(x)−d_(p)) that ispresent in this embodiment.

Suitable ranges for particularly preferred embodiments of the inventionas shown in the figures are provided below:

Post diameter range (d_(p)): 10 nm-5 mm;

Number of columns (n_(c)): 1-10,000;

Number of rows (n_(r)): 3-10,000;

Gap range (g): 10 nm-5 mm;

Shift (s): 0-5 mm;

Column pitch (p_(c)): 30 nm 50 mm; and

Row pitch (p_(r)) 30 nm-50 mm

The disclosure of every patent, patent application, and publicationcited herein is hereby incorporated herein by reference in its entirety.

The citation of any reference herein should not be construed as anadmission that such reference is available as “Prior Art” to the instantapplication.

Throughout the specification the aim has been to describe the preferredembodiments of the invention without limiting the invention to any oneembodiment or specific collection of features. Those of skill in the artwill therefore appreciate that, in light of the instant disclosure,various modifications and changes can be made in the particularembodiments exemplified without departing from the scope of theinvention. All such modifications and changes are intended to beincluded within the scope of the appended claims.

What is claimed is:
 1. A method for introducing an exogenous materialinto a cell, comprising: introducing a liquid including the cell and theexogenous material into a flow channel of a microfluidic device, thechannel including at least two flow diverters, the gap between the atleast two flow diverters having a width, the width of the gap beinggreater than the diameter of the cell; and exposing said cell to atransient decrease in pressure and unsteady flow downstream of the flowdiverters when the cell flows past the flow diverters to therebyintroduce said exogenous material into said cell.
 2. The method of claim1, wherein the cell is viable after being exposed to the transientdecrease in pressure and unsteady flow.
 3. The method of claim 1,wherein said exogenous material is selected from the group consisting ofa small organic molecule, a nucleic acid, a nucleotide, anoligonucleotide, a protein, a peptide, an amino acid, a lipid, apolysaccharide, a quantum dot, a carbon nanotube, a nanoparticle, a goldparticle, a monosaccharide, a vitamin and a steroid.
 4. The method ofclaim 1, wherein said exogenous material is introduced into thecytoplasm of said cell.
 5. The method of claim 1, wherein said cell is amammalian cell, a yeast cell or an insect cell and said exogenousmaterial is introduced into the nucleus of said cell.
 6. The method ofclaim 1, wherein said cell is exposed to said transient decrease inpressure in the presence of said exogenous material for at least 10nanoseconds.
 7. The method of claim 1, wherein unsteady flow is at leastone of a laminar vortex street, a transitional vortex street, aturbulent vortex street, transitional flow or turbulent flow.
 8. Amethod for introducing an exogenous material into a cell, comprising:introducing a liquid including the cell and the exogenous material intoa flow channel of a microfluidic device, the channel including at leasttwo flow diverters, the gap between the at least two flow divertershaving a width, the width of the gap being greater than the diameter ofthe cell; and exposing said cell to a transient decrease in pressure andunsteady flow downstream of the flow diverters when the cell flows pastthe flow diverters to thereby introduce said exogenous material intosaid cell, wherein said transient decrease in pressure is at least 10kPa.
 9. The method of claim 1, wherein each flow diverter is a post. 10.The method of claim 9, wherein the post is cylindrical.
 11. The methodof claim 10, wherein the post has a diameter greater than the gapbetween the posts.
 12. The method of claim 10, wherein the post has adiameter equal to the gap between the posts.
 13. The method of claim 10,wherein the post has a diameter less than the gap between the posts. 14.The method of claim 1, wherein each flow diverter has a maximum width of10 micrometers.
 15. The method of claim 1, wherein each flow diverterhas a maximum width of 100 micrometers.
 16. The method of claim 1,wherein each flow diverter has a maximum width of 1 millimeter.
 17. Amethod for transfecting a cell to introduce an exogenous material intothe cell, comprising: introducing a liquid including the cell and theexogenous material into a flow channel of a microfluidic device, theflow channel having a width and at least two flow diverters oriented inan array along the width of the flow channel, each flow diverter havinga maximum width, the maximum width of each flow diverter being greaterthan the gap between the at least two flow diverters; and exposing thecell to a transient decrease in pressure and unsteady flow downstream ofthe flow diverters when the cell flows past the flow diverters tothereby introduce the exogenous material into the cell.
 18. The methodof claim 17, wherein the at least two flow diverters include a pluralityof posts.
 19. The method of claim 17, wherein the maximum width of eachflow diverter is 10 micrometers.
 20. The method of claim 17, whereineach flow diverter has a height greater than its maximum width.
 21. Themethod of claim 1, wherein the exogenous material introduced into thecell is an expression vector.
 22. The method of claim 21, wherein theexpression vector is a bacterial artificial chromosome (BAC).
 23. Themethod of claim 1, wherein the cell is a mammalian cell having a nucleusand the exogenous material is introduced into the nucleus.
 24. Themethod of claim 1, wherein the cell is exposed to the transient decreasein pressure and unsteady flow in the presence of the exogenous materialfor at least 10 nanoseconds.
 25. The method of claim 1, wherein the flowhas a maximum peak velocity of at least 1 meter per second.
 26. Themethod of claim 1, wherein the flow of the liquid around the flowdiverters has an object Reynolds number of at least
 40. 27. The methodof claim 17, wherein the cell is exposed to the transient decrease inpressure and unsteady flow in the presence of the exogenous material forat least 10 nanoseconds.
 28. The method of claim 17, wherein the flowhas a maximum peak velocity of at least 1 meter per second.
 29. Themethod of claim 17, wherein the flow of the liquid around the flowdiverters has an object Reynolds number of at least
 40. 30. The methodof claim 8, wherein the flow of the liquid around the flow diverters hasan object Reynolds number of at least 40.